Cells respond to and interact with their environments through their plasma membrane (PM), thus the PM is a critical interface for cell physiology. The proteins in the PM provide the basis for these physiological responses; accordingly, cells have elaborate mechanisms to regulate which proteins are found in specific cellular domains. Temporal control is also important, in order to effect rapid or sustained changes, as needed. One very important mechanism for controlling the composition of the PM is endocytosis, a pathway for the selective removal of membrane proteins and lipids. Endocytosis is also important for bringing essential nutrients into the cell and for retrieving intracellular proteins that are transiently incorporated into the PM. Adaptor proteins select specific membrane cargo for internalization. Scaffolding proteins are thought to promote efficiency of the process by coordinating cargo collection with formation of the endocytic structure and ultimately, separation of the endocytic vesicle. Adaptors and scaffolds must be regulated in order to turn on and off as needed. In this proposal, we will examine the functions and regulation of Pan1, an evolutionarily conserved scaffolding protein in the budding yeast Saccharomyces cerevisiae. We will test a model in which phosphorylation and the sequential recruitment of binding partners regulates a cycle of interactions between adaptors, scaffolds, and actin-effectors that may underlie the transitions from 'early to late to completed' endocytic events. Understanding the mechanisms of this fundamental cell regulatory pathway has wide-spread implications: many viruses and pathogens gain entry into cells via endocytosis; cancer cells can proliferate due to an overabundance of signaling receptors that have not been properly internalized; and gene therapy strategies make use of endocytosis as a way to deliver the active compounds into the cell.